Jeffrey T. Kroetsch

Sphingosine-1-Phosphate–Dependent Activation of p38 MAPK Maintains Elevated Peripheral Resistance in Heart Failure Through Increased Myogenic Vasoconstriction

Rationale: Mechanisms underlying vasomotor abnormalities and increased peripheral resistance exacerbating heart failure (HF) are poorly understood. Objective: To explore the role and molecular basis of myogenic responses in HF. Methods and Results: 10 weeks old C57Bl6 mice underwent experimental myocardial infarction (MI) or sham surgery. At 1 to 12 weeks postoperative, mice underwent hemodynamic studies, mesenteric, cerebral, and cremaster artery perfusion myography and Western blot. Organ weights and hemodynamics confirmed HF and increased peripheral resistance after MI. Myogenic responses, ie, pressure-induced vasoconstriction, were increased as early as 1 week after MI and remained elevated. Vasoconstrictor responses to phenylephrine were decreased 1 week after MI, but not at 2 to 6 weeks after MI, whereas those to endothelin (ET)-1 and sphingosine-1-phosphate (S1P) were increased at all time points after MI. An antagonist (JTE-013) for the most abundant S1P receptor detected in mesenteric arteries (S1P2R) abolished the enhanced myogenic responses of HF, with significantly less effect on controls. Mice with genetic absence of sphingosine-kinases or S1P2R (Sphk1−/−; Sphk1−/−/Sphk2+/−; S1P2R−/−) did not manifest enhanced myogenic responses after MI. Mesenteric arteries from HF mice exhibited increased phosphorylation of myosin light chain, with deactivation of its phosphatase (MLCP). Among known S1P-responsive regulators of MLCP, GTP-Rho levels were unexpectedly reduced in HF, whereas levels of activated p38 MAPK and ERK1/2 (extracellular signal-regulated kinase 1/2) were increased. Inhibiting p38 MAPK abolished the myogenic responses of animals with HF, with little effect on controls. Conclusions: Rho-independent p38 MAPK-mediated deactivation of MLCP underlies S1P/S1P2R-regulated increases in myogenic vasoconstriction observed in HF. Therapeutic targeting of these findings in HF models deserves study.

Tumor Necrosis Factor-α–Mediated Downregulation of the Cystic Fibrosis Transmembrane Conductance Regulator Drives Pathological Sphingosine-1-Phosphate Signaling in a Mouse Model of Heart Failure

Background— Sphingosine-1-phosphate (S1P) signaling is a central regulator of resistance artery tone. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzyme sphingosine kinase 1 and its functional antagonist S1P phosphohydrolase-1. The intracellular localization of S1P phosphohydrolase-1 necessitates the import of extracellular S1P into the intracellular compartment before its degradation. The present investigation proposes that the cystic fibrosis transmembrane conductance regulator transports extracellular S1P and hence modulates microvascular S1P signaling in health and disease. Methods and Results— In cultured murine vascular smooth muscle cells in vitro and isolated murine mesenteric and posterior cerebral resistance arteries ex vivo, the cystic fibrosis transmembrane conductance regulator (1) is critical for S1P uptake; (2) modulates S1P-dependent responses; and (3) is downregulated in vitro and in vivo by tumor necrosis factor-&agr;, with significant functional consequences for S1P signaling and vascular tone. In heart failure, tumor necrosis factor-&agr; downregulates the cystic fibrosis transmembrane conductance regulator across several organs, including the heart, lung, and brain, suggesting that it is a fundamental mechanism with implications for systemic S1P effects. Conclusions— We identify the cystic fibrosis transmembrane conductance regulator as a critical regulatory site for S1P signaling; its tumor necrosis factor-&agr;–dependent downregulation in heart failure underlies an enhancement in microvascular tone. This molecular mechanism potentially represents a novel and highly strategic therapeutic target for cardiovascular conditions involving inflammation.

Nitric oxide and coronary vascular endothelium adaptations in hypertension

This review highlights a number of nitric oxide (NO)-related mechanisms that contribute to coronary vascular function and that are likely affected by hypertension and thus become important clinically as potential considerations in prevention, diagnosis, and treatment of coronary complications of hypertension. Coronary vascular resistance is elevated in hypertension in part due to impaired endothelium-dependent function of coronary arteries. Several lines of evidence suggest that other NO synthase isoforms and dilators other than NO may compensate for impairments in endothelial NO synthase (eNOS) to protect coronary artery function, and that NO-dependent function of coronary blood vessels depends on the position of the vessel in the vascular tree. Adaptations in NOS isoforms in the coronary circulation to hypertension are not well described so the compensatory relationship between these and eNOS in hypertensive vessels is not clear. It is important to understand potential functional consequences of these adaptations as they will impact the efficacy of treatments designed to control hypertension and coronary vascular disease. Polymorphisms of the eNOS gene result in significant associations with incidence of hypertension, although mechanistic details linking the polymorphisms with alterations in coronary vasomotor responses and adaptations to hypertension are not established. This understanding should be developed in order to better predict those individuals at the highest risk for coronary vascular complications of hypertension. Greater endothelium-dependent dilation observed in female coronary arteries is likely related to endothelial Ca2+ control and eNOS expression and activity. In hypertension models, the coronary vasculature has not been studied extensively to establish mechanisms for sex differences in NO-dependent function. Genomic and nongenomic effects of estrogen on eNOS and direct and indirect antioxidant activities of estrogen are discussed as potential mechanisms of interest in coronary circulation that could have implications for sex- and estrogen status-dependent therapy for hypertension and coronary dysfunction. The current review identifies some important basic knowledge gaps and speculates on the potential clinical relevance of hypertension adaptations in factors regulating coronary NO function.

Proximal Cerebral Arteries Develop Myogenic Responsiveness in Heart Failure via Tumor Necrosis Factor-α–Dependent Activation of Sphingosine-1-Phosphate Signaling

Background— Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Methods and Results— Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-&agr; (TNF&agr;)–dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNF&agr; expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNF&agr; antagonist etanercept. TNF&agr; mediates its effect via a sphingosine-1-phosphate (S1P)–dependent mechanism, requiring sphingosine kinase 1 and the S1P2 receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Conclusions— Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNF&agr;/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Proximal Cerebral Arteries Develop Myogenic Responsiveness in Heart Failure via TNFα-Dependent Activation of S1P Signaling

Background— Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Methods and Results— Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-&agr; (TNF&agr;)–dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNF&agr; expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNF&agr; antagonist etanercept. TNF&agr; mediates its effect via a sphingosine-1-phosphate (S1P)–dependent mechanism, requiring sphingosine kinase 1 and the S1P2 receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Conclusions— Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNF&agr;/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries

Objective—The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results—In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1S225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations, and Ca2+ sensitivity. The lack of function of the Sk1S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca2+ sensitivity and myogenic tone were attenuated, whereas Ca2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca2+ sensitivity) and intracellular effects of S1P (Ca2+ handling). Accordingly, S1P2 receptor inhibition (1&mgr;mol/L JTE013) attenuated myogenic tone without effect on Ca2+. Conclusions—Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.

Disrupting the Key Circadian Regulator CLOCK leads to Age-Dependent Cardiovascular Disease.

The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in heart weight, hypertrophy, dilation, impaired contractility, and reduced myogenic responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts. Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3β, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3β, S6K1 phosphorylation are increased, consistent with the development of age-dependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in growth and renewal; disrupting Clock leads to age-dependent cardiomyopathy. Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

The TNF-α/Sphingosine-1-Phosphate Signaling Axis Drives Myogenic Responsiveness in Heart Failure

Heart failure (HF) is hallmarked by an increase in total peripheral resistance (TPR) that compensates for the drop in cardiac output. While initially allowing for the maintenance of mean arterial pressure at acceptable levels, the long-term upregulation of TPR is prone to compromise cardiac performance and tissue perfusion, and to ultimately accelerate disease progression. Augmented vasoconstriction of terminal arteries, the site of TPR regulation, is cooperatively driven by mechanisms such as: (i) endothelial dysfunction, (ii) increased sympathetic activity and (iii) enhanced pressure-induced myogenic responsiveness. Herein, we review emerging evidence that the increase in myogenic responsiveness is central to the long-term elevation of TPR in HF. On a molecular level, this augmented intrinsic response is governed by an activation of the tumor necrosis factor-α (TNF-α)/sphingosine-1-phosphate signaling axis in microvascular smooth muscle cells. The beneficial effect of TNF-α scavenging strategies on tissue perfusion in HF mouse models adds to the gaining momentum to revisit the use of anti-TNF-α treatment modalities in discrete HF patient populations.

Tumor necrosis factor / sphingosine-1-phosphate signaling augments resistance artery myogenic tone in diabetes

Diabetes strongly associates with microvascular complications that ultimately promote multiorgan failure. Altered myogenic responsiveness compromises tissue perfusion, aggravates hypertension, and sets the stage for later permanent structural changes to the microcirculation. We demonstrate that skeletal muscle resistance arteries isolated from patients with diabetes have augmented myogenic tone, despite reasonable blood glucose control. To understand the mechanisms, we titrated a standard diabetes mouse model (high-fat diet plus streptozotocin [HFD/STZ]) to induce a mild increase in blood glucose levels. HFD/STZ treatment induced a progressive myogenic tone augmentation in mesenteric and olfactory cerebral arteries; neither HFD nor STZ alone had an effect on blood glucose or resistance artery myogenic tone. Using gene deletion models that eliminate tumor necrosis factor (TNF) or sphingosine kinase 1, we demonstrate that vascular smooth muscle cell TNF drives the elevation of myogenic tone via enhanced sphingosine-1-phosphate (S1P) signaling. Therapeutically antagonizing TNF (etanercept) or S1P (JTE013) signaling corrects this defect. Our investigation concludes that vascular smooth muscle cell TNF augments resistance artery myogenic vasoconstriction in a diabetes model that induces a small elevation of blood glucose. Our data demonstrate that microvascular reactivity is an early disease marker and advocate establishing therapies that strategically target the microcirculation.

Cerebral artery myogenic reactivity: The next frontier in developing effective interventions for subarachnoid hemorrhage:

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating cerebral event that kills or debilitates the majority of those afflicted. The blood that spills into the subarachnoid space stimulates profound cerebral artery vasoconstriction and consequently, cerebral ischemia. Thus, once the initial bleeding in SAH is appropriately managed, the clinical focus shifts to maintaining/improving cerebral perfusion. However, current therapeutic interventions largely fail to improve clinical outcome, because they do not effectively restore normal cerebral artery function. This review discusses emerging evidence that perturbed cerebrovascular “myogenic reactivity,” a crucial microvascular process that potently dictates cerebral perfusion, is the critical element underlying cerebral ischemia in SAH. In fact, the myogenic mechanism could be the reason why many therapeutic interventions, including “Triple H” therapy, fail to deliver benefit to patients. Understanding the molecular basis for myogenic reactivity changes in SAH holds the key to develop more effective therapeutic interventions; indeed, promising recent advancements fuel optimism that vascular dysfunction in SAH can be corrected to improve outcome.